Contrast-Enhanced Microtomographic Characterisation of Vessels in Native Bone and Engineered Vascularised Grafts Using Ink-Gelatin Perfusion and Phosphotungstic Acid

Bone ischemia and necrosis are challenging to treat, requiring investigation of native and engineered bone revascularisation processes through advanced imaging techniques. This study demonstrates an experimental two-step method for precise bone and vessel analysis in native bones or vascularised bone grafts using X-ray microtomography (; μ; CT), without interfering with further histological processing.; Distally ligated epigastric arteries or veins of 6 nude rats were inserted in central channels of porous hydroxyapatite cylinders and these pedicled grafts were implanted subcutaneously. One week later, the rats were perfused with ink-gelatin and euthanised and the femurs, tibias, and grafts were explanted. Samples were scanned using; μ; CT, decalcified, incubated with phosphotungstic acid (PTA) for contrast enhancement, rescanned, and processed histologically.; Contrast-enhanced; μ; CT displayed the course and branching of native bone vessels. Histologically, both central (-17%) and epiphyseal vessels (-58%) appeared smaller than in; μ; CT scans. Hydroxyapatite cylinders were thoroughly vascularised but did not display bone formation. Grafts with a central artery had more (+58%) and smaller (-52%) vessel branches compared to grafts with a vein.; We present a relatively inexpensive and easy-to-perform two-step method to analyse bone and vessels by; μ; CT, suitable to assess a variety of bone-regenerative strategies.